Use of biguanide derivatives for making a medicine having a wound healing effect

ABSTRACT

The present invention relates to the use of biguanide derivatives of general, formula I below;  
                 
 
     in which:  
     the groups R1 and R2 represent, independently of each other, a hydrogen atom, a C 1 -C 7 , alkyl group, a cycloalkyl group, a heterocycle, a C 2 -C 7  alkenyl group, an aryl group, an aralkyl group, an aryloxyalkyl group or a heteroaryl group,  
     or R1 and R2, taken together, represent a C 2 -C 7  alkylene which may contain one or more hetero atoms,  
     and the group R3 represents a primary, secondary or tertiary amine,  
     or pharmaceutically acceptable salts thereof, to manufacture a medicinal product with cicatrizing action, the said medicinal product being in a pharmaceutical form for topical use.

[0001] The present invention relates to the cicatrization of wounds. Theinvention relates in particular to the use of biguanide derivatives orpharmaceutically acceptable salts thereof, advantageously metformin, tomanufacture a medicinal product with cicatrizing action.

[0002] The cicatrization of wounds or similar injuries on tissues ofvarious type generally depends on the proliferation of newtissues—epithelial, endothelial and connective. It thus involves aseries of co-ordinated cellular and molecular events. It may be delayedor modified by metabolic disturbances which accompany certainlong-lasting diseases such as venous insufficiency, arteritis, diabetesand even certain therapies.

[0003] The pharmaceutical market currently offers many topicalpreparations recommended for cicatrizing wounds. In point of fact, theiraction results from the complementarity of the various products of whichthey are composed and which give them, to a certain extent, theircicatrizing properly. They protect wounds from the surrounding medium byan antiseptic dressing. They stimulate the development ofviscularization and regulate epidermization. These topical forms consistmainly of a lipid mixture (lanolin, petroleum jelly, glycerol, etc.) towhich are added acids (salicylic acid, benzoic acid, malic acid),minerals (zinc oxide, titanium oxide) or halides (starch iodide).

[0004] Some preparations also contain collagen, fibrinogen, enzymaticserum proteolysate (supply of amino acids) or vitamins (vitamin A) orhormones (4-chloro-testosterone acetate). There is also an ointment(Madecasol® tulgras from the Laboratoires Syntex), the cicatrizatingaction of which is provided by the addition of a mixture of threetriterpenes extracted from the roots of the plant Centella asiatica(TCEA). These compounds exert their property by stimulating thebiosynthesis of collagen and glycoaminoglycans. However, these extractsmay also cause contact allergies in patients.

[0005] It is known that one of the complications of diabetes is theappearance of skin complaints such as ulcers (or even ulcerous necroticangiodermatitis) or perforating dermatoses which the conventionalmedicinal products used in diabetes treatments do not manage either tocontrol or to treat.

[0006] Pharmaceutical compositions based on biguanides are also alreadyknown. However, they are only used in the treatment of certain forms ofdiabetes, and mainly of non-insulin-dependant type II diabetes, asanti-hyperglycaemiant agents which promote the return to glycaemicequilibrium. Metformin is the biguanide derivative most frequently usedin this type of treatment.

[0007] The daily dosage is between 500 mg and 3 g depending on thediabetic's degree of glycaemia. Metformin has a high therapeutic marginin man and is considered as a medicinal product that is well-tolerated.

[0008] The anti-hyperglycaemic effect of metformin is thought to be duefirstly to the increase in the endogenous insulin activity and secondlyto the action of metformin via insulin-independent mechanisms.Specifically, the action of metformin is reflected by a decrease in theintestinal absorption of glucose, an increase in cellular absorption ofblood glucose and a decrease in glucose production by the liver(suppression of neoglucogenesis) and also the amount of insulin requiredto normalize glycaemia. These effects result partly from the power ofmetformin to amplify the action of the existing insulin by increasingthe activity of the enzyme tyrosine kinase of the insulin receptor,which triggers the “post-receptor” signal cascade.

[0009] Patent application FR 2 213 778 discloses novel compositions fortreating proliferative skin diseases, which may contain a biguanidederivative: phenformin. Proliferative skin diseases are benign andmalignant skin diseases which are characterized by a chronic excessiveproliferation of the cells of the epidermis, of the dermis or of theirinclusions. The compositions disclosed in this patent application reducethis excessive proliferation and therefore do not have a cicatrizingaction, that is to say an acceleration of tissue growth.

[0010] Now, the inventors of the present invention have revealed,surprisingly, that biguanide derivatives and in particular metforminalso have strong cicatrizing properties, that is to say stimulatoryactivity towards a complex physiological phenomenon characterized, interalia, by increased cell growth in the region of the wound. Thistransient proliferation arises in response to the loss of skin integrityand ensures repair of the deep tissue and reconstitution of theepidermis in the region of the wounds.

[0011] Thus, the topical application of this compound in the form of anointment induces rapid and long-lasting healing of leg ulcers indiabetic individuals and repetition of the topical application of theactive principle reinforces this effect. Furthermore, metformin alsoaccelerates the cicatrization of atonic wounds in non-diabeticindividuals.

[0012] Given the difficulties encountered to control the quality ofnatural cicatrizing products and the number of laborious steps requiredto isolate these compounds, biguanide derivatives, whose synthesis issimple, total and rapid, appear to be highly advantageous activeprinciples.

[0013] The present invention thus relates to the use of biguanidederivatives of general formula I below:

[0014] in which:

[0015] the groups R1 and R2 represent, independently of each other, ahydrogen atom, a C₁-C₇ alkyl group, a cycloalkyl group, a heterocycle, aC₂-C₇ alkenyl group, an aryl group, an aralkyl group, an aryloxyalkylgroup or a heteroaryl group,

[0016] or R1 and R2, taken together, represent a C₂-C₇ alkylene whichmay contain one or more hetero atoms,

[0017] and the group R3 represents a primary, secondary or tertiaryamine,

[0018] or pharmaceutically acceptable salts thereof, to manufacture amedicinal product with cicatrizing action, the said medicinal productbeing in a pharmaceutical form for topical use. This medicinal productis advantageously intended to be applied to the skin.

[0019] For the purposes of the present invention, the expression “C₁-C₇alkyl group” means any linear or branched, substituted or unsubstitutedC₁-C₇ alkyl group, such as, for example, methyl, ethyl, propyl,isopropyl or butyl groups and also isomers thereof.

[0020] For the purposes of the present invention, the expression“cycloalkyl group” means any cycloalkyl group containing from 3 to 7carbon atoms, such as, for example, the cyclohexyl group.

[0021] For the purposes of the present invention, the term “heterocycle”means any ring containing from 3 to 7 atoms, one or more of which beinga hetero atom such as, for example, a nitrogen, oxygen or sulphur atom,the others being carbon atoms.

[0022] For the purposes of the present invention, the expression “C₂-C₇alkenyl group” means any linear or branched, substituted orunsubstituted C₂-C₇ alkenyl group, such as vinyl or allyl groups.

[0023] For the purposes of the present invention, the expression “arylgroup” means any hydrocarbon-based aromatic group such as, for example,the phenyl group, which may contain one or more substituents, such as,for example, a C₁-C₇ alkyl group as defined above, a C₂-C₇ alkenyl groupas defined above or a halogen.

[0024] For the purposes of the present invention, the expression“aralkyl group” means any aryl group as defined above linked via analkyl as defined above. Advantageously, when the alkyl group representsCH₂ and the aryl group represents a phenyl group, this phenyl group issubstituted in the manner defined above, and when the alkyl group doesnot represent CH₂, the aryl group is as defined above, advantageously aphenyl group.

[0025] For the purposes of the present invention, the expression“aryloxyalkyl group” means any aryl group as defined above linked via anoxyalkyl group whose alkyl residue is as defined above.

[0026] For the purposes of the present invention, the expression“heteroaryl group” means any hydrocarbon-based aromatic group containingone or more hetero atoms, such as, for example, sulphur, nitrogen oroxygen atoms, and which can bear one or more substituents, such as, forexample, a C₁-C₇ alkyl group as defined above, a C₂-C₇ alkenyl group asdefined above or a halogen. Examples of heteroaryl groups are furyl,isoxazyl, pyridyl and pyrimidyl groups.

[0027] For the purposes of the present invention, the expression “C₂-C₇alkylene group” means any C₂-C₇ alkylene group such as, for example,ethylene, trimethylene, tetramethylene or pentamethylene groups.

[0028] For the purposes of the present invention, the expression“pharmaceutically acceptable salt” means any salt prepared from anypharmaceutically acceptable non-toxic acid, including organic acids andmineral acids. Such acids include acetic acid, benzenesulphonic acid,benzoic acid, citric acid, ethanesulphonic acid, fumaric acid, gluconicacid, glutamic acid, hydrobromic acid, hydrochloric acid, lactic acid,maleic acid, malic acid, mandelic acid, methanesulphonic acid, mucicacid, nitric acid, palmoic acid, paintothenic acid, phosphoric acid,succinic acid, tartaric acid and para-toluenesulphonic acid.Hydrochloric acid is advantageously used.

[0029] For the purposes of the present invention, the expression“pharmaceutical form for topical use” means any pharmaceutical formintended to be applied to the surface of the wound, in particular to theskin or external or internal mucous membranes, and which acts locally.In particular, the medicinal product may be in a form such as an oil,cream, mousse, liniment, lotion, ointment, liquid, gel, milk, powder orspray. The forms may contain a one-phase vehicle and may consist of aneutral hydroxypropylcellulose gel or a charged gel formed from sodiumcarboxymethylcellulose. It is also possible to prepare creams, which areforms containing a two-phase vehicle, comprising a hydrophilic phasedispersed in a lipophilic phase. The medicinal product is advantageouslyin the form of a gel or an ointment. The medicinal product mayadvantageously be in the form of an active dressing, the said dressingconsisting of a support on which the biguanide derivative(s) is(are)impregnated or supported, advantageously in the form of a gel or anointment. In particular, the active dressing consists of the combinationof a hydrocolloid dressing and one or more biguanide derivatives.

[0030] In one particular embodiment of the invention, the groups R1 andR2 represent, independently of each other, a hydrogen atom, a C₁-C₇alkyl group, a cycloalkyl group, a heterocycle, a C₂-C₇ alkenyl group,an aryloxyalkyl group or a heteroaryl group.

[0031] In another particular embodiment of the invention, the group R3represents a secondary amine of the following formula:

[0032] Advantageously, the biguanide derivative used is metformin, evenmore advantageously in the form of a hydrochloride.

[0033] In one particular example, the medicinal product contains from0.02% to 2% by weight of a biguanide derivative of general formula I orthe pharmaceutically acceptable salt thereof and a suitable excipient.These excipients may be chosen from compounds with good compatibilitywith these active principles. They are, for example, water-solublepolymers of natural polymer type, such as polysaccharides (xanthan gum,carob gum, peptin, etc.) or polypeptides, cellulose derivatives such asmethylcellulose, hydroxypropylcellulose or hydroxypropylmethylcellose,or alternatively synthetic polymers, polaxamers, carbomers, PVA or PVP.

[0034] Finally, it is within the competence of a person skilled in theart to add to these medicinal products various excipients, for exampleco-solvents, for instance ethanol, glycerol or benzyl alcohol, wettingagents (glycerol), agents for facilitating diffusion (transcurol, urea)or antibacterial preserving agents (0.15% methyl p-hydroxybenzoate).

[0035] In one particular embodiment of the invention, the biguanidederivatives or pharmaceutically acceptable salts thereof are combinedwith at least one other active principle. This active principle may be,for example, of the antibiotic, antifungal or antiviral agent type, thusmaking it possible to accelerate the cicatrization of damaged andinfected tissues, simultaneously or in combination with the treatment ofthe underlying infection.

[0036] This active principle may also consist of another agent forimproving cicatrization such as, for example, epidermal growth factor,fibroblast growth factor, platelet-derived growth factor, etc.

[0037] The biguanide derivatives of general formula I and thepharmaceutically acceptable salts thereof, in particular metformin,advantageously in hydrochloride form, can thus improve the cicatrizationof wounds or lesions of any type. These wounds or lesions may be of thetype such as surgical incisions, thermal or chemical burns, burns causedby irradiation, abrasions, lacerations, amputations, ischaemic ulcers orbedsores, oral lesions or ulcers or corneal lesions, and in particularthose caused by surgery performed on run-down individuals, the elderly,individuals treated by radiotherapy or chemotherapy, or diabetics. Thisis likewise the case for all dermatoses observed in patients whosecutaneous circulation is deficient (erythemal lesions, vascularitis) andfor all wounds observed in diabetic individuals. The pharmaceuticalcompositions and medicinal products according to the invention alsoappear to be beneficial in the treatment of tissue necrosis, for examplepost-thrombotic tissue necrosis.

[0038] The examples below of compositions according to the invention aregiven by way of illustration and with no limiting nature.

EXAMPLES

[0039] Several pharmaceutical forms were prepared without preservingagent. The percentages are expressed on a weight basis.

Formulation Example 1

[0040] Metformin: 1% by weight relative to the weight of gel.

[0041] Neutral gel of hydroxypropylcellulose (Klucel from Aqualon, type99 MF EP) at 2.9%: remainder to 100%.

Formulation Example 2

[0042] Metformin 1% by weight relative to the weight of gel.

[0043] Charged gel of sodium carboxymethylcellulose (Aqualon) at 4.5%:remainder to 100%.

Formulation Example 3

[0044] Metformin: 1% by weight relative: to the lipophilic phase.

[0045] Hydrocorin emulsion (fatty excipient from Roc® containingpetroleum jelly, liquid paraffin, triglycerides, polyoxyethylene ethersand ceresin) at 33% (H/L: hydrophilic phase dispersed in a lipophilicphase): remainder to 100%.

[0046] This emulsion is prepared at 73° C. by pouring the water in whichthe metformin has been dissolved into the fatty phase and stirring untilcool.

[0047] Other subjects and advantages of the invention will becomeapparent to a person skilled in the art from the detailed descriptionbelow and by reference to the following illustrative drawings.

[0048]FIG. 1 represents the effect of an ointment according toFormulation Example 3 comprising metformin at 1% by weight relative tothe lipophilic phase, on cutaneous cicatrization.

[0049]FIG. 2 represents the “dose-response” study of an ointmentaccording to Formulation Example 3 comprising metformin at differentconcentrations, on cutaneous cicatrization.

[0050]FIG. 3 represents the relative surface area occupied by thebundles of collagen in the granulation tissue at D4 according to thepercentage of metformin present in the ointment according to FormulationExample 3.

[0051] The efficacy of metformin was tested in vivo on skin woundsreproducing ulceration. The pseudo-ulcer was produced on 14-week-oldZucker fa/fa rats by loss of matter of circular shape 8 mm in diametermade by a “punch”, down to the muscle layer. The daily treatment of thewound with metformin contained in an ointment according to FormulationExample 3 at different metformin concentrations systematically led to asignificant improvement in cicatrization. The speed of the cicatrizationwas evaluated by determining the area of the wounds with a video cameracoupled to a computer equipped with image analysis software.

[0052] The treatment of the wounds for 10 days (D0-D10) with theointment according to Formulation Example 3 comprising metformin at aconcentration of 1% by weight relative to the lipophilic phase made itpossible macroscopically to show a large reduction in the area of thetreated wounds relative to the control wounds (FIG. 1). The microscopicevaluation of the cicatrization at D5 and D10 indicates an increase inthe quality of the scar tissue in the wounds treated with the ointmentaccording to Formulation Example 3 comprising metformin (more developedgranulation tissue, more advanced re-epidermization). Furthermore,metformin at a concentration of 1% by weight relative to the lipophilicphase also appears to exert an effect on neovascularizaton. This effectbecomes evident at D5 via a greater density of blood capillaries on theedges of the wound for the treated rats relative to the control rats.

[0053] The results of a “dose-response” comparative study carried outwith an ointment according to Formulation Example 3 comprising metforminat concentrations of 0.02%, 0.1%, and 2% by weight relative to thelipophilic phase reveal cicatrizing action for all the concentrationstested. For the four metformin concentrations used, the closure of thetreated wounds is faster than of the untreated control wounds (FIG. 2).

[0054] The results of the microscopic analysis of histological sectionsof the wounds are collated in Table 1 below. TABLE 1 Effect of differentweight concentrations of metformin relative to the lipophilic phase inan ointment according to Formulation Example 3 on the formation ofepidermis (biopsy taken from one third of the wound) Control D4 D6 D8D10 1 − − ± + 2 − − + + 3 − − + + 4 − ± + + 5 − + + + 6 − + + + Relativeaverage  0%  50% 100% 100% Ointment comprising 0.02% metformin D4 D6 D8D10 1 ± + + + 2 − + + + 3 + + + + 4 − + + + Relative average 50% 100%100% 100% Ointment comprising 0.1% metformin D4 D6 D8 D10 1 − + + + 2− + + + 3 − + + + 4 − + + + Relative average  0% 100% 100% 100% Ointmentcomprising 1% metformin D4 D6 D8 D10 1 − + + + 2 − ± + + 3 + ± + + 4− + + + Relative average 25% 100% 100% 100% Ointment comprising 2%metformin D4 D6 D8 D10 1 − + + + 2 − − − + 3 − + + + 4 ± ± ± + Relativeaverage 25%  75%  75% 100%

[0055] These results clearly indicate an acceleration of epidermizationin animals treated with metformin at the different concentrationscompared with untreated control animals.

[0056] Moreover, the treatment of wounds with metformin significantlyincreases the speed of maturation of granulation tissue, which isreflected, inter alia, by an increase in the collagen density (FIG. 3).

1. Use of biguanide derivatives of general formula I below:

in which; the groups R1 and R2 represent, independently of each other, ahydrogen atom, a C₁-C₇ alkyl group, a C₃-C₇ cycloalkyl group, aheterocycle, a C₂-C₇ alkenyl group, an aryl group, an aralkyl group thealkyl group of which is C₁-C₇, an aryloxyalkyl group the alkyl group ofwhich is C₁-C₇ or a heteroaryl group, or R1 and R2, taken together,represent a C₂-C₇ alkylene which may contain one or more hetero atoms,and the group R3 represents a primary or tertiary amine, or an amine offormula:

or pharmaceutically acceptable salts thereof, to manufacture a medicinalproduct with cicatrizing action, the said medicinal product being in apharmaceutical form for topical use.
 2. Use according to claim 1, tomanufacture a medicinal product with cicatrizing action, in apharmaceutical form for topical use intended to be applied to the skin,advantageously in the form of an ointment, optionally supported orimpregnated on a dressing so as to constitute an active dressing.
 3. Useaccording to either of the preceding claims, to manufacture a medicinalproduct with cicatrizing action on the wounds of diabetic individuals.4. Use according to any one of the preceding claims characterized inthat the groups R1 and R2 represent, independently of each other, ahydrogen atom, a C₁-C₇ alkyl group, a C₃-C₇ cycloalkyl group, aheterocycle, a C₂-C₇ alkenyl group, an aryloxyalkyl group the alkylgroup of which is C₁-C₇ or a heteroaryl group.
 5. Use according to anyone of the preceding claims, characterized in that the biguanidederivative used is metformin, advantageously in the form of ahydrochloride.
 6. Use according to any one of the preceding claims,characterized in that the biguanide derivatives or pharmaceuticallyacceptable salts thereof are combined with at least one other activeprinciple.